GSM8084230: DRIPc-TGFb; Mus musculus; OTHER
DNA was extracted by treating cell samples with SDS and Proteinase K at 37ºC overnight, followed by phenol-chloroform isoamyl alcohol extraction using phase lock gel tubes (VWR, Cat #: 10847-800) followed by ethanol precipitation. Extracted DNA was digested with restriction enzyme cocktail (BsrGI (NEB, Cat #: R3575S), EcoRI (NEB, Cat #: R3101S), HindIII (NEB, Cat #: R3104S), SspI (NEB, Cat #: R3132S), XbaI (NEB, Cat #: R0145S)) in NEB buffer 2 with 1 mM spermidine (Sigma Aldrich, Cat #: 05292-1ML-F) and bovine serum albumin (BSA) (NEB, Cat #: B9001) at 37ºC overnight. Digested DNAs were purified by phenol-chloroform isoamyl alcohol (Thermo Fisher Scientific, Cat #: 15593031) extraction using phase lock gel tube followed by ethanol precipitation. 10 μg of digested DNA were incubated with RNase H (NEB, Cat #: M0297S) for 6 h at 37ºC in RNase H buffer. 8 μg of DNA/RNA hybrids treated with RNase H and 8 μg of DNA/RNA hybrids from five separate tubes without RNase H were immunoprecipitated with S9.6 antibodies (Millipore Sigma, Cat #: MABE1095) at 4ºC overnight on rotator. Dynabeads™ Protein G beads were blocked with 2.5 mg/mL UltraPure™ BSA (Thermo Scientific) at 4ºC overnight. The beads were then washed twice with binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl and 0.05% Triton X-100, 10 mM Tris-HCl pH 8 and 1 mM EDTA) and incubated with the DNA/RNA-antibody samples at 4ºC for 2 h. The beads were washed three times with binding buffer, followed by incubation with 300 μL of elution buffer and 7 μL of proteinase K at 55ºC for 45 min with mixing. After incubation, DNA/RNA hybrids were separated by magnetic rack and subjected to Phenol-Chloroform Isoamyl alcohol extraction using phase lock gel tubes followed by ethanol precipitation. Recovered DNA/RNA hybrids were incubated with DNase I (NEB, Cat #: M0303S) at 37ºC for 45 min and DNase I was then inactivated by addition of 1 μL of 0.5 M EDTA, pH 8.0 and incubation at 75ºC for 15 min. Ethanol-precipitated RNA strands were subjected to reverse transcription with IScript reverse transcription supermix (BioRad, Cat #: 1708840) per manufacturer's instructions. cDNA samples were cleaned by AMPure XP beads to recover DNA fragments 200-500 bp. Eluted fragments were subjected to second strand synthesis with dUTP followed by AMPure XP bead purification. Samples were subjected to sonication using a Diagenode Bioruptor with 15 cycles, end repair (NEB, Cat #: E6050), dATP-tailing (NEB, Cat #: M0212S), and adaptor ligation with TruSeq Single indexes (Illumina, Cat #: 20015960). Libraries then cleared of uracil-containing DNA strands using uracil N-glycosylase (Thermo Fisher Scientific, Cat #: N8080096), amplified, and purified with AMPure XP beads.